Proteasomes, the effectors of the ubiquitin-proteasome system that controls protein degradation, are structurally and functionally complex molecular machines containing more than 30 distinct proteins. Selective degradation of labile regulatory proteins is essential to cellular regulation; proteasomes also eliminate potentially toxic misfolded proteins. The misregulation of proteolysis is implicated in a broad array of disease mechanisms, and the proteasome has been validated as a target of cancer chemotherapy. The overall goal of this project is to understand better the mechanism of proteasome substrate degradation. Detailed mechanistic understanding of proteasome action requires more fully defining the intermediate states in this multi-step process and understanding how interactions among the different proteins and subassemblies of the proteasome are coordinated and participate in substrate processing. We will make use of a set of substrates with distinct requirements for proteasomal degradation. These require progressively restricted subsets of proteasome functions for their recognition and/or degradation, as follows: Ubiquitinated substrates: Proteasome must perform all intrinsic functions. Substrates bearing ubiquitin-independent degradation tag: Ubiquitin control functions redundant. Substrates tethered to proteasome: Capture redundant, downstream events required. The following Specific Aims will be carried out in furtherance of those goals: Aim 1. Determine how the proteasome recognizes and captures an ubiquitin-independent degron Biochemical and genetic strategies will be used to identify the receptor for the degron, the degradation signal, of ornithine decarboxylase. Aim 2. Define whether and how substrate mechanical stability affects processing time and ATP consumption Representatives of each of the three classes described above will be used to investigate the interactions among processing kinetics, substrate mechanical stability and ATP hydrolysis. Aim 3. Determine the pathway of a substrate through the proteasome and changes in subunit contacts during its processing. We will use the substrates described above to examine how proteasome components and substrates change their interactions during processing. Aim 4. Perturb the substrate processing pathway by means of site-directed proteasome mutations We will use defined proteasome mutations, including those of ATPases and the alpha ring gate, to alter the trajectory of degradation, thereby facilitating the detection and ordering of intermediate states within the multi-step pathway PUBLIC HEALTH RELEVANCE: Cells must recognize and destroy certain of their protein components- those that are aberrant or have completed a programmed task. The engine of destruction is termed the proteasome. This large complex encompasses about 30 different kinds of proteins and must coordinates its multiple enzyme activities so as to first recognize proteins marked for destruction and finally reduce them to small fragments. We are proposing to study the series of biochemical events that underlie this processing pathway. The system which specifies protein destruction is mis-regulated in a broad array of disease, including cancer, and the proteasome has been validated as a target of cancer chemotherapy.